Large-plaque mutants of Sindbis virus show reduced binding to heparan sulfate, heightened viremia, and slower clearance from the circulation

Abstract

Laboratory strains of Sindbis virus must bind to the negatively charged glycosaminoglycan heparan sulfate in order to efficiently infect cultured cells. During infection of mice, however, we have frequently observed the development of large-plaque viral mutants with a reduced ability to bind to heparan sulfate. Sequencing of these mutants revealed changes of positively charged amino acids in putative heparin-binding domains of the E2 glycoprotein. Recombinant viruses were constructed with these changes as single amino acid substitutions in a strain Toto 1101 background. All exhibited decreased binding to heparan sulfate and had larger plaques than Toto 1101. When injected subcutaneously into neonatal mice, large-plaque viruses produced higher-titer viremia and often caused higher mortality. Because circulating heparin-binding proteins are known to be rapidly sequestered by tissue heparan sulfate, we measured the kinetics of viral clearance following intravenous injection. Much of the parental small-plaque Toto 1101 strain of Sindbis virus was cleared from the circulation by the liver within minutes, in contrast to recombinant large-plaque viruses, which had longer circulating half-lives. These findings indicate that a decreased ability to bind to heparan sulfate allows more efficient viral production in vivo, which may in turn lead to increased mortality. Because Sindbis virus is only one of a growing number of viruses from many families which have been shown to bind to heparan sulfate, these results may be generally applicable to the pathogenesis of such viruses.

FIG. 1

Viral binding to heparin. Radiolabeled SV was applied to heparin-Sepharose and eluted with a 50 to 500 mM NaCl gradient. Elution at a higher NaCl concentration indicates stronger binding to heparin. Eluted virus is intact and fully infectious. (Five other viruses are not shown; see Table 2 for results.)

FIG. 2

Viral binding to cultured cells. Radiolabeled SV was allowed to bind to CHO or glycosaminoglycan-deficient pgsA-745 cells at 4°C for 2 h. The mean binding ± standard deviation for three replicates is shown. The data along the x axis are taken from Table 2.

FIG. 3

Kinetics of viral clearance. Mice were injected intravenously with a 100-μl bolus of purified virus. Serum was collected, and titers were determined by a plaque assay with BHK cells at various times postinjection; V/V₀ indicates the fraction of virus remaining. Each point represents the mean ± standard deviation for three mice. Curves were fitted by nonlinear regression as described in Materials and Methods. Symbols: ●, K76T; ○, K230M; ▾, N62D; ▿, K159E; ■, K76E; □, K76N; ⧫, R157H; ◊, Toto 1101.

FIG. 4

Serum titers. (A) CD-1 pups (2 days old) were infected subcutaneously with 1,000 PFU of virus, and serum titers were assessed at various times by a plaque assay (mean for three mice at each time point; error bars are not shown). All LP viruses retained their LP phenotype. A two-way analysis of variance showed a significant (P, <0.0001) effect of viral strain on titer, and titers of Toto 1101 were lower than those of all seven mutant viruses when compared by Scheffe's test (P, <0.05). (B) Plotting of peak titer (mean ± standard deviation) versus elution from heparin-Sepharose failed to indicate a simple correlation between virus replication in vivo and the strength of binding to heparin.

FIG. 5

Survival curves. Litters of 2-day-old CD-1 pups were infected subcutaneously with 1,000 PFU of virus, and survival was monitored for 15 days. R157H, K230M, N62D, and K76E produced significantly greater mortality than Toto 1101. Survival curves were compared pairwise against Toto 1101 by use of the log rank test with a threshold for significance (P) of <0.0071; the Bonferonni correction for multiple comparisons was used.